Pyrrolopyrimidine compounds and uses thereof for modulating glucocerebrosidase activity

ABSTRACT

Disclosed are new small molecules having a pyrrolopyrimidine core structure and the uses thereof for modulating glucocerebrosidase activity. Also disclosed are pharmaceutical compositions comprising the small molecules which may be administered in methods of treating diseases or disorders associated with glucocerebrosidase activity, includin neurological diseases and disorders such as Gaucher&#39;s disease and Parkinson&#39;s disease. The small molecules may be utilized to generate activated glucocerebrosidase. The activated glucocerebrosidase thusly generated can be administered in enzyme replacement therapy and/or utilized in screening assays for new small molecules that bind to the activated glucocerebrosidase and/or modulate the activity of the activated glucocerebrosidase.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to and the benefit of U.S. PatentApplication No. 62/725,830, filed on Aug. 31, 2018, the entire contentsof which are herein incorporated by reference.

FIELD

The field of the invention relates to new small molecules and uses ofthe new small molecules for modulating glucocerebrosidase (GCase)activity such as β-GCase activity. The new small molecules have apyrrolopyrimidine core structure, such as apyrrrolo[1,2-a]pyrimidine-8-carboxamide core structure, and the smallmolecules may be administered to treat diseases and disorders associatedwith aberrant glucocerebrosidase activity including, but not limited toneurodegenerative diseases, such as Gaucher's disease and Parkinson'sdisease.

BACKGROUND

Glucocerebrosidase (EC 3.2.1.45), which also is calledβ-glucocerebrosidase, (β-glucosidase, D-glucosyl-N-acylsphingosineglucohydrolase, or GCase, is an enzyme having glucosylceramidaseactivity. Glucocerebrosidase is required to cleave the beta-glucosidiclinkage of the chemical glucocerebroside, which is an intermediate inglycolipid metabolism. Glucocerebrosidase is localized in the lysosomeand disabling mutations in the gene for glucocerebrosidase (GBA1) areassociated with abnormal accumulation of lipids in lysosomes includingglucosylceramide (GL1).

Genetic diseases caused by mutations in GBA1 include neurodegenerativediseases such as Gaucher's disease and Parkinson's disease. Gaucher'sdisease is a rare genetic disease caused by GBA1 gene mutations.Currently, the treatment for Type 1 Gaucher's disease is enzymereplacement therapy (ERT) in which a modified form of glucocerebrosidaseis administered intravenously every two weeks in order to reduce orprevent the buildup of lipids including GL1 in lysosomes. ERT is veryexpensive and not effective for neuronopathic forms of Gaucher'sdisease. Mutations in GBA1 also are linked to Parkinson's disease (PD)and are associated with an increased risk of PD.

SUMMARY

The present disclosure provides novel substituted pyrrolopyrimidinecompounds which modulate glucocerebrosidase activity. The substitutedpyrrolopyrimidine compounds disclosed herein have better chemical andphysical properties than previous reported non-active site GCaseinhibitors. (See Goldin et al., WO, “Substituted pyrazolopyrimidines asglucocerebrosidase activators.” December 2010, WO2012078855; and Patnaiket al., “Discovery, structure-activity relationship, and biologicalevaluation of noninhibitory small molecule chaperones ofglucocerbrosidase,” J. Med. Chem. 2012 Jun. 28;55(1{grave over( )}2):5734-48, the contents of which are incorporated herein byreference in their entireties). These better chemical and physicalproperties of the disclosed substituted pyrrolopyrimidine compoundsinclude larger polar surface area, increased solubility, increasednumber of rotatable bonds, and increased number of potential hydrogenbonding members. The chemical and physical properties of the disclosedsubstituted pyrrolopyrimidine compounds also better meet therequirements for oral bioavailability and blood-brain penetration.

Some of the substituted pyrrolopyrimidine compounds in the presentdisclosure are capably of highly activating GCase. For example, some ofthe substituted pyrrolopyrimidine compounds bind to GCase covalently andactivate wild-type GCase up to 70-100 fold. GCase thusly activated bythe disclosed pyrrolopyrimidine compounds is observed to be more stablein an acidic environment than GCase that has not been activated.Therefore, when used in enzyme replacement therapy, the activated GCaselikely can be administered at a lower dose and less frequently thanGCase that has not been activated by the disclosed compounds.

The activated GCase disclosed herein also can be used in a GCaseactivity assay for screening compounds that bind and/or modulate theenzyme activity of GCase. The disclosed GCase activity assay is veryuseful to identify new compounds having a binding affinity and/ormodulatory activity for GCase. No high throughput screening method haspreviously been developed based on such a binding affinity assay foractivated GCase.

Some embodiments of the present disclosure provide new, small moleculeshaving a substituted pyrrolopyryimidine core structure, in particular, a4-methylpyrrrolo[1,2-a]pyrimidine-8-carboxamide core structure. The new,small molecules may be utilized for modulating glucocerebrosidaseactivity. The new, small molecules preferably modulateglucocerebrosidase activity by binding to glucocerebrosidase, optionallycovalently, and activating glucocerebrosidase. The new small moleculesmay be formulated as pharmaceutical compositions that comprise the smallmolecules or that comprise activated glucocerebrosidase conjugated tothe small molecules, which compositions may be administered in methodsof treating and/or preventing diseases or disorders associated withglucocerebrosidase activity, including neurological diseases anddisorders such as Gaucher's disease and Parkinson's disease. Theactivated glucocerebrosidase also may be utilized in screening methodsfor test compounds to identify new compounds that bind to activatedglucocerebrosidase and/or that modulate the activity of activatedglucocerebrosidase.

In some embodiments, the present disclosure provides a compound or asalt or solvate thereof having a Formula I:

wherein:

-   m is 0-6;-   is selected from

-   n is 0-6;-   p is 0-6;-   R² is C1-C6 alkyl or pyridinyl;-   R³ is selected from hydrogen, C1-C6 alkyl, C1-C6 alkoxy, phenyl,    nitrophenyl, phenoxy, amino, —C(O)—O—R¹², —N—C(O)—R¹³, and

-   R⁴ is —(OCH₂CH₂)_(q)— where q is 0-6;-   R⁵ is hydrogen, C1-C6 alkyl, or C3-C6 cycloalkyl;-   R⁶ and R⁷ are independent selected from hydrogen and C1-C6 alkyl;-   R⁸ is hydrogen, C1-C6 alkyl, phenyl, or benzyl;-   R⁹ is hydrogen, C1-C6 alkyl, or C1-C6 dialkyl;-   R¹⁰ and R¹¹ are independently selected from hydrogen, C1-C6 alkyl,    phenyl, and benzyl;-   R¹² is selected from hydrogen, C1-C6 alkyl, phenyl, and    N-succinimidyl; and-   R¹³ is selected from hydrogen, C1-C6 alkyl, C1-C6 (halo)alkyl, and    C2-C6 alkenyl.

In further embodiments, the compound has a Formula Ia:

In some embodiments, the compound has a formula of:

In further embodiments, the compound has a Formula Ib:

In some embodiments, the compound has a formula selected from:

In further embodiments, R³ in any one of the preceding compounds isselected from —C(O)—O—R¹² and —N—C(O)—R¹³.

In some embodiments, the compound has a formula:

In further embodiments, the compound has a formula:

Some embodiments of the present disclosure provide a pharmaceuticalcomposition comprising any one of the preceding compounds and apharmaceutical carrier.

Further embodiments of the present disclosure provide a method fortreating a disease or disorder that is associated withglucocerebrosidase activity in a subject in need thereof, the methodcomprising administering the pharmaceutical composition having any oneof the preceding compounds and the pharmaceutical carrier.

In some embodiments, the disease or disorder is a neurological diseaseor disorder.In further embodiments, the degenerative neurologicaldisease or disorder is Gaucher's disease.In some embodiments, thedegenerative neurological disease or disorder is Parkinson's disease.

In further embodiments, any one of the preceding compounds is covalentlyattached to glucocerebrosidase to form a conjugate. The conjugate thusformed may be formulated as a pharmaceutical composition whichoptionally is administered to a subject in need thereof

Some embodiments of the present disclosure provide a method of screeningfor a test compound having binding activity or modulatory activity forglucocerebrosidase, the method comprising: (a) contacting the testcompound with an activated glucocerebrosidase that has been activated bycovalently attaching to the glucocerebrosidase a compound according toany of the preceding formulas, and (b) detecting enzyme activity of theactivated glucocerebrosidase in the presence of the test compound.

In further embodiments, detecting enzyme activity comprises (i)contacting the activated glucocerebrosidase and the test compound with asubstrate that is metabolized by the activated glucocerebrosidase togenerate a fluorescent metabolite; and (ii) detecting fluorescence ofthe fluorescent metabolite.

DETAILED DESCRIPTION

The disclosed subject matter further may be described utilizing terms asdefined below.

Unless otherwise specified or indicated by context, the terms “a”, “an”,and “the” mean “one or more.” For example, “a modulator ofglucocerebrosidase activity” should be interpreted to mean “one or moremodulators of glucocerebrosidase activity.”

As used herein, “about”, “approximately,” “substantially,” and“significantly” will be understood by persons of ordinary skill in theart and will vary to some extent on the context in which they are used.If there are uses of the term which are not clear to persons of ordinaryskill in the art given the context in which it is used, “about” and“approximately” will mean plus or minus ≤10% of the particular term and“substantially” and “significantly” will mean plus or minus >10% of theparticular term.

As used herein, the terms “include” and “including” have the samemeaning as the terms “comprise” and “comprising.” The terms “comprise”and “comprising” should be interpreted as being “open” transitionalterms that permit the inclusion of additional components further tothose components recited in the claims. The terms “consist” and“consisting of” should be interpreted as being “closed” transitionalterms that do not permit the inclusion additional components other thanthe components recited in the claims. The term “consisting essentiallyof” should be interpreted to be partially closed and allowing theinclusion only of additional components that do not fundamentally alterthe nature of the claimed subject matter.

The terms “subject,” “patient,” and “individual” may be usedinterchangeably herein. A subject may be a human subject. A subject mayrefer to a human subject having or at risk for acquiring a disease ordisorder that is associated with aberrant glucocerebrosidase activity.As used herein, the term “aberrant” means higher or lower activityrelative to a normal healthy subject. In specific embodiments, a subjectexhibiting aberrant glucocerebrosidase may have or be at risk foracquiring Gaucher's disease and/or neurological diseases and disorderssuch as genetic and sporadic synucleinopathies, including Parkinson'sdisease, dementia with Lewy bodies, and multiple system atrophyassociated with aberrant glucocerebrosidase activity.

As used herein, the term “modulate” means decreasing or inhibitingactivity and/or increasing or augmenting activity. For example,modulating glucocerebrosidase activity may mean increasing or augmentingglucocerebrosidase activity and/or decreasing or inhibitingglucocerebrosidase activity. The compounds disclosed herein may beadministered to modulate glucocerebrosidase activity for example, as achaperone or activator.

As used herein, the phrase “effective amount” shall mean that drugdosage that provides the specific pharmacological response for which thedrug is administered in a significant number of patients in need of suchtreatment. An effective amount of a drug that is administered to aparticular patient in a particular instance will not always be effectivein treating the conditions/diseases described herein, even though suchdosage is deemed to be a therapeutically effective amount by those ofskill in the art.

New Chemical Entities

New chemical entities and uses for chemical entities are disclosedherein. The chemical entities may be described using terminology knownin the art and further discussed below.

As used herein, an asterick “*” or a plus sign “+” may be used todesignate the point of attachment for any radical group or substituentgroup.

The term “alkyl” as contemplated herein includes a straight-chain orbranched alkyl radical in all of its isomeric forms, such as a straightor branched group of 1-12, 1-10, or 1-6 carbon atoms, referred to hereinas C1-C12 alkyl, C1-C10-alkyl, and C1-C6-alkyl, respectively.

The term “alkylene” refers to a diradical of an alkyl group (e.g.,—(CH₂)_(n)— where n is an integer such as an integer between 1 and 20).An exemplary alkylene group is —CH₂CH₂—.

The term “haloalkyl” refers to an alkyl group that is substituted withat least one halogen. For example, —CH₂F, —CHF₂, —CF₃, —CH₂CF₃, —CF₂CF₃,and the like.

The term “heteroalkyl” as used herein refers to an “alkyl” group inwhich at least one carbon atom has been replaced with a heteroatom(e.g., an O, N, or S atom). One type of heteroalkyl group is an “alkoxy”group.

The term “alkenyl” as used herein refers to an unsaturated straight orbranched hydrocarbon having at least one carbon-carbon double bond, suchas a straight or branched group of 2-12, 2-10, or 2-6 carbon atoms,referred to herein as C2-C12-alkenyl, C2-C10-alkenyl, and C2-C6-alkenyl,respectively.

The term “alkynyl” as used herein refers to an unsaturated straight orbranched hydrocarbon having at least one carbon-carbon triple bond, suchas a straight or branched group of 2-12, 2-10, or 2-6 carbon atoms,referred to herein as C2-C12-alkynyl, C2-C10-alkynyl, and C2-C6-alkynyl,respectively.

The term “cycloalkyl” refers to a monovalent saturated cyclic, bicyclic,or bridged cyclic (e.g., adamantyl) hydrocarbon group of 3-12, 3-8, 4-8,or 4-6 carbons, referred to herein, e.g., as “C4-8-cycloalkyl,” derivedfrom a cycloalkane. Unless specified otherwise, cycloalkyl groups areoptionally substituted at one or more ring positions with, for example,alkanoyl, alkoxy, alkyl, haloalkyl, alkenyl, alkynyl, amido, amidino,amino, aryl, arylalkyl, azido, carbamate, carbonate, carboxy, cyano,cycloalkyl, ester, ether, formyl, halo, haloalkyl, heteroaryl,heterocyclyl, hydroxyl, imino, ketone, nitro, phosphate, phosphonato,phosphinato, sulfate, sulfide, sulfonamido, sulfonyl or thiocarbonyl. Incertain embodiments, the cycloalkyl group is not substituted, i.e., itis unsubstituted.

The term “cycloalkylene” refers to a cycloalkyl group that isunsaturated at one or more ring bonds.

The term “partially unsaturated carbocyclyl” refers to a monovalentcyclic hydrocarbon that contains at least one double bond between ringatoms where at least one ring of the carbocyclyl is not aromatic. Thepartially unsaturated carbocyclyl may be characterized according to thenumber of ring carbon atoms. For example, the partially unsaturatedcarbocyclyl may contain 5-14, 5-12, 5-8, or 5-6 ring carbon atoms, andaccordingly be referred to as a 5-14, 5-12, 5-8, or 5-6 memberedpartially unsaturated carbocyclyl, respectively. The partiallyunsaturated carbocyclyl may be in the form of a monocyclic carbocycle,bicyclic carbocycle, tricyclic carbocycle, bridged carbocycle,spirocyclic carbocycle, or other carbocyclic ring system. Exemplarypartially unsaturated carbocyclyl groups include cycloalkenyl groups andbicyclic carbocyclyl groups that are partially unsaturated. Unlessspecified otherwise, partially unsaturated carbocyclyl groups areoptionally substituted at one or more ring positions with, for example,alkanoyl, alkoxy, alkyl, haloalkyl, alkenyl, alkynyl, amido, amidino,amino, aryl, arylalkyl, azido, carbamate, carbonate, carboxy, cyano,cycloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl,heterocyclyl, hydroxyl, imino, ketone, nitro, phosphate, phosphonato,phosphinato, sulfate, sulfide, sulfonamido, sulfonyl or thiocarbonyl. Incertain embodiments, the partially unsaturated carbocyclyl is notsubstituted, i.e., it is unsubstituted.

The term “aryl” is art-recognized and refers to a carbocyclic aromaticgroup. Representative aryl groups include phenyl, naphthyl, anthracenyl,and the like. The term “aryl” includes polycyclic ring systems havingtwo or more carbocyclic rings in which two or more carbons are common totwo adjoining rings (the rings are “fused rings”) wherein at least oneof the rings is aromatic and, e.g., the other ring(s) may becycloalkyls, cycloalkenyls, cycloalkynyls, and/or aryls. Unlessspecified otherwise, the aromatic ring may be substituted at one or morering positions with, for example, halogen, azide, alkyl, aralkyl,alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro,sulfhydryl, imino, amido, carboxylic acid, —C(O)alkyl, —CO₂alkyl,carbonyl, carboxyl, alkylthio, sulfonyl, sulfonamido, sulfonamide,ketone, aldehyde, ester, heterocyclyl, aryl or heteroaryl moieties,—CF₃, —CN, or the like. In certain embodiments, the aromatic ring issubstituted at one or more ring positions with halogen, alkyl, hydroxyl,or alkoxyl. In certain other embodiments, the aromatic ring is notsubstituted, i.e., it is unsubstituted. In certain embodiments, the arylgroup is a 6-10 membered ring structure.

The terms “heterocyclyl” and “heterocyclic group” are art-recognized andrefer to saturated, partially unsaturated, or aromatic 3- to 10-memberedring structures, alternatively 3-to 7-membered rings, whose ringstructures include one to four heteroatoms, such as nitrogen, oxygen,and sulfur. The number of ring atoms in the heterocyclyl group can bespecified using 5 Cx-Cx nomenclature where x is an integer specifyingthe number of ring atoms. For example, a C3-C7 heterocyclyl group refersto a saturated or partially unsaturated 3- to 7-membered ring structurecontaining one to four heteroatoms, such as nitrogen, oxygen, andsulfur. The designation “C3-C7” indicates that the heterocyclic ringcontains a total of from 3 to 7 ring atoms, inclusive of any heteroatomsthat occupy a ring atom position.

The terms “amine” and “amino” are art-recognized and refer to bothunsubstituted and substituted amines (e.g., mono-substituted amines ordi-substituted amines), wherein substituents may include, for example,alkyl, cycloalkyl, heterocyclyl, alkenyl, and aryl.

The terms “alkoxy” or “alkoxyl” are art-recognized and refer to an alkylgroup, as defined above, having an oxygen radical attached thereto.Representative alkoxy groups include methoxy, ethoxy, tert-butoxy andthe like.

An “ether” is two hydrocarbons covalently linked by an oxygen.Accordingly, the substituent of an alkyl that renders that alkyl anether is or resembles an alkoxyl, such as may be represented by one of-O-alkyl, -O-alkenyl, -O-alkynyl, and the like.

The term “carbonyl” as used herein refers to the radical —C(O)—.

The term “oxo” refers to a divalent oxygen atom —O—.

The term “carboxamido” as used herein refers to the radical —C(O)NRR′,where R and R′ may be the same or different. R and R′, for example, maybe independently alkyl, aryl, arylalkyl, cycloalkyl, formyl, haloalkyl,heteroaryl, or heterocyclyl.

The term “carboxy” as used herein refers to the radical —COOH or itscorresponding salts, e.g. —COONa, etc.

The term “amide” or “amido” or “amidyl” as used herein refers to aradical of the form —R¹C(O)N(R²)—, —R¹C(O)N(R²)R³, —C(O)NR²R³, or—C(O)NH₂, wherein R¹, R² and R³, for example, are each independentlyalkoxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl,carbamate, cycloalkyl, ester, ether, formyl, halogen, haloalkyl,heteroaryl, heterocyclyl, hydrogen, hydroxyl, ketone, or nitro.

The compounds of the disclosure may contain one or more chiral centersand/or double bonds and, therefore, exist as stereoisomers, such asgeometric isomers, enantiomers or diastereomers. The term“stereoisomers” when used herein consist of all geometric isomers,enantiomers or diastereomers. These compounds may be designated by thesymbols “R” or “S,” or “+” or “−” depending on the configuration ofsubstituents around the stereogenic carbon atom and or the opticalrotation observed. The present invention encompasses various stereoisomers of these compounds and mixtures thereof. Stereoisomers includeenantiomers and diastereomers. Mixtures of enantiomers or diastereomersmay be designated (±)” in nomenclature, but the skilled artisan willrecognize that a structure may denote a chiral center implicitly. It isunderstood that graphical depictions of chemical structures, e.g.,generic chemical structures, encompass all stereoisomeric forms of thespecified compounds, unless indicated otherwise. Also contemplatedherein are compositions comprising, consisting essentially of, orconsisting of an enantiopure compound, which composition may comprise,consist essential of, or consist of at least about 50%, 60%, 70%, 80%,90%, 95%, 96%, 97%, 98%, 99%, or 100% of a single enantiomer of a givencompound (e.g., at least about 99% of an R enantiomer of a givencompound).

Pyrrolopyrimidine Compounds

The compounds disclosed herein may be referred to as pyrrolopyrimidinecompounds. In particular, the compounds disclosed herein by be referredto as pyrrolo[1,2-a]pyrimidine compounds. The disclosed compounds mayinclude one or more substituents at one or more positions of thepyrrolo[1,2-a]pyrimidine core of the compound. In some embodiments, thepyrrolo[1,2-a]pyrimidine compounds disclosed herein may be referred toas “4-methylpyrrrolo[1,2-a]pyrimidine-8-carboxamide compounds.”

The compounds or salt or solvates thereof may be described as having aFormula I as follows:

wherein:

m is 0-1, -2, -3, -4, -5, or -6;

R¹ is selected from

n is 0-1, -2, -3, -4, -5, or -6;

p is 0-1, -2, -3, -4, -5, or -6;

R² is C1-C6 alkyl or pyridinyl (e.g., pyridin-2-yl, pyridin-3-yl, orpyridin-4-yl);

R³ is selected from hydrogen, C1-C6 alkyl, C1-C6 alkoxy, phenyl,nitrophenyl (e.g., 4-nitrophenyl), phenoxy, amino, *—C(O)—O—R¹²,*—N—C(O)—R¹³, and

R⁴ is —(OCH₂CH₂)_(q)— where q is 0-1, -2, -3, -4, -5, or -6;

R⁵ is hydrogen, C1-C6 alkyl, or C3-C6 cycloalkyl;

R⁶ and R⁷ are independent selected from hydrogen and C1-C6 alkyl;

R⁸ is hydrogen, C1-C6 alkyl, phenyl, or benzyl;

R⁹ is hydrogen, C1-C6 alkyl (e.g., methyl), or C1-C6 dialkyl (e.g.,dimethyl);

R¹⁰ and R¹¹ are independently selected from hydrogen, C1-C6 alkyl (e.g.,isobutyl), phenyl, and benzyl;

R¹² is selected from hydrogen, C1-C6 alkyl, phenyl, and N-succinimidyl;and

R¹³ is selected from hydrogen, C1-C6 alkyl, C1-C6 (halo)alkyl, and C2-C6alkenyl.

In some embodiments, the pyrrolo[1,2-a]pyrimidine compounds disclosedherein may be referred to as“2,4-dimethylpyrrrolo[1,2-a]pyrimidine-8-carboxamide compounds,” havinga Formula Ia as follows:

where R¹ is as defined above for Formula I.

The disclosed compounds may include two substituted4-methylpyrrrolo[1,2-a]pyrimidine-8-carboxamide groups conjugated via alinker. Compounds that include two substituted4-methylpyrrrolo[1,2-a]pyrimidine-8-carboxamide groups conjugated via alinker between the carboxamide groups may be illustrated as follows:

In some embodiments, the disclosed compounds having two substituted4-methylpyrrrolo[1,2-a]pyrimidine-8-carboxamide groups conjugated via alinker optionally where the linker has a formula—(CH₂)_(v)-phenyl-(CH₂)_(w)— and v and w are independently selected from0-1, 0-2, 0-3, 0-4, 0-5, or 0-6. In particular, the disclosed compoundshaving two substituted 4-methylpyrrrolo[1,2-a]pyrimidine-8-carboxamidegroups conjugated via a linker may be described as having a formula asfollows:

As noted, the compounds disclosed herein, including the substitutedpyrimidine and fused pyrimidine compounds discussed above may haveseveral chiral centers, and stereoisomers, epimers, and enantiomers arecontemplated. The compounds may be optically pure with respect to one ormore chiral centers (e.g., some or all of the chiral centers may becompletely in the S configuration; some or all of the chiral centers maybe completely in the R configuration; etc.). Additionally oralternatively, one or more of the chiral centers may be present as amixture of configurations (e.g., a racemic or another mixture of the Rconfiguration and the S configuration). Compositions comprisingsubstantially purified stereoisomers, epimers, or enantiomers, oranalogs or derivatives thereof are contemplated herein (e.g., acomposition comprising at least about 90%, 95%, 99% or 100% purestereoisomer, epimer, or enantiomer.) As used herein, formulae which donot specify the orientation at one or more chiral centers are meant toencompass all orientations and mixtures thereof.

The disclosed compounds may comprise or may be conjugated to afluorophore. For example, the disclosed compounds may be conjugated tofluorophore via a linker (i.e., compound-linker-fluorophore). Suitablelinkers may include but are not limited to a linker having a formula—NH—C(O)—(CH₂)_(p)—NH—C(O)—(CH₂CH₂O)_(q)—CH₂—CH₂—NH—C(O)— where p and qare independently selected from 1-18, 1-12, or 1-6 (e.g., where p=5 andq=4)). In some embodiments of the disclosed compounds of Formula I orIa, any of substituents R¹, R², R³, R⁴, R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁,R₁₂, and R¹³, may comprise a fluorophore and/or may be conjugated to afluorophore optionally via a linker, including fluorophores suitable foruse in fluorescence polarization assays.

As used herein, a “fluorophore” is a chemical group that can be excited(e.g., by light or a chemical reaction) to emit fluorescence. Somesuitable fluorophores may be excited by light to emit phosphorescence.As used herein, a “dye” may include a fluorophore. The compoundsdescribed herein may be conjugated to a fluorophore selected from butnot limited to: 1,5 IAEDANS; 1,8-ANS; 4-Methylumbelliferone;5-carboxy-2,7-dichlorofluorescein; 5-Carboxyfluorescein (5-FAM); 5-Carboxytetramethylrhodamine (5 -TAMRA); 5 -FAM (5-Carboxyfluorescein);5-HAT (Hydroxy Tryptamine); 5-Hydroxy Tryptamine (HAT); 5-ROX(carboxy-X-rhodamine); 5 -TAMRA (5 -Carboxytetramethylrhodamine);6-Carboxyrhodamine 6G; 6-CR 6G; 6-JOE; 7-Amino-4-methylcoumarin;7-Aminoactinomycin D (7-AAD); 7-Hydroxy-4-methylcoumarin;9-Amino-6-chloro-2-methoxyacridine; ABQ; Acid Fuchsin; ACMA(9-Amino-6-chloro-2-methoxyacridine); Acridine Orange; Acridine Red;Acridine Yellow; Acriflavin; Acriflavin Feulgen SITSA; Alexa Fluor 350™;Alexa Fluor 430™; Alexa Fluor 488™; Alexa Fluor 532™; Alexa Fluor 546™;Alexa Fluor 568™; Alexa Fluor 594™; Alexa Fluor 633™; Alexa Fluor 647™;Alexa Fluor 660™; Alexa Fluor 680™; Alizarin Complexon; Alizarin Red;Allophycocyanin (APC); AMC; AMCA-S; AMCA (Aminomethylcoumarin); AMCA-X;Aminoactinomycin D; Aminocoumarin; Aminomethylcoumarin (AMCA); AnilinBlue; Anthrocyl stearate; APC (Allophycocyanin); APC-Cy7; APTS; AstrazonBrilliant Red 4G; Astrazon Orange R; Astrazon Red 6B; Astrazon Yellow 7GLL; Atabrine; ATTO-TAG™ CBQCA; ATTO-TAG™ FQ; Auramine; Aurophosphine G;Aurophosphine; BAO 9 (Bisaminophenyloxadiazole); Berberine Sulphate;Beta Lactamase; BFP blue shifted GFP (Y66H); Blue Fluorescent Protein;BFP/GFP FRET; Bimane; Bisbenzamide; Bisbenzimide (Hoechst); BlancophorFFG; Blancophor SV; BOBO™-1; BOBO™-3; Bodipy 492/515; Bodipy 493/503;Bodipy 500/510; Bodipy 505/515; Bodipy 530/550; Bodipy 542/563; Bodipy558/568; Bodipy 564/570; Bodipy 576/589; Bodipy 581/591; Bodipy630/650-X; Bodipy 650/665-X; Bodipy 665/676; Bodipy FL; Bodipy FL ATP;Bodipy Fl-Ceramide; Bodipy R₆G SE; Bodipy TMR; Bodipy TMR-X conjugate;Bodipy TMR-X, SE; Bodipy TR; Bodipy TR ATP; Bodipy TR-X SE; BO-PRO™-1;BO-PRO™-3; Brilliant Sulphoflavin FF; Calcein; Calcein Blue; CalciumCrimson™; Calcium Green; Calcium Orange; Calcofluor White;Carboxy-X-rhodamine (5-ROX); Cascade Blue™; Cascade Yellow;Catecholamine; CCF2 (GeneBlazer); CFDA; CFP—Cyan Fluorescent Protein;CFP/YFP FRET; Chlorophyll; Chromomycin A; CL-NERF (Ratio Dye, pH);CMFDA; Coelenterazine f; Coelenterazine fcp; Coelenterazine h;Coelenterazine hcp; Coelenterazine ip; Coelenterazine n; CoelenterazineO; Coumarin Phalloidin; C-phycocyanine; CPM Methylcoumarin; CTC; CTCFormazan; Cy2™; Cy3.1 8; Cy3.5™; Cy3™; Cy5.1 8; Cy5.5™; Cy5™; Cy7™; CyanGFP; cyclic AMP Fluorosensor (FiCRhR); Dabcyl; Dansyl; Dansyl Amine;Dansyl Cadaverine; Dansyl Chloride; Dansyl DHPE; Dansyl fluoride; DAPI;Dapoxyl; Dapoxyl 2; Dapoxyl 3; DCFDA; DCFH (DichlorodihydrofluoresceinDiacetate); DDAO; DHR (Dihydorhodamine 123); Di-4-ANEPPS; Di-8-ANEPPS(non-ratio); DiA (4-Di-16-ASP); Dichlorodihydrofluorescein Diacetate(DCFH); DiD—Lipophilic Tracer; DiD (DiIC18(5)); DIDS; Dihydorhodamine123 (DHR); DiI (DiIC18(3)); Dinitrophenol; DiO (DiOC18(3)); DiR; DiR(DiIC18(7)); DNP; Dopamine; DsRed; DTAF; DY-630-NHS; DY-635-NHS; EBFP;ECFP; EGFP; ELF 97; Eosin; Erythrosin; Erythrosin ITC; Ethidium Bromide;Ethidium homodimer-1 (EthD-1); Euchrysin; EukoLight; Europium (III)chloride; EYFP; Fast Blue; FDA; Feulgen (Pararosaniline); FITC; FlazoOrange; Fluo-3; Fluo-4; Fluorescein (FITC); Fluorescein Diacetate;Fluoro-Emerald; Fluoro-Gold (Hydroxystilbamidine); Fluor-Ruby; FluorX;FM 1-43™; FM 4-46; Fura Red™; Fura Red™/Fluo-3; Fura-2; Fura-2/BCECF;Genacryl Brilliant Red B; Genacryl Brilliant Yellow 10GF; Genacryl Pink3G; Genacryl Yellow 5GF; GeneBlazer (CCF2); GFP (S65T); GFP red shifted(rsGFP); GFP wild type, non-UV excitation (wtGFP); GFP wild type, UVexcitation (wtGFP); GFPuv; Gloxalic Acid; Granular Blue;Haematoporphyrin; Hoechst 33258; Hoechst 33342; Hoechst 34580; HPTS;Hydroxycoumarin; Hydroxystilbamidine (FluoroGold); Hydroxytryptamine;Indo-1; Indodicarbocyanine (DiD); Indotricarbocyanine (DiR); IntrawhiteCf; JC-1; JO-JO-1; JO-PRO-1; Laurodan; LDS 751 (DNA); LDS 751 (RNA);Leucophor PAF; Leucophor SF; Leucophor WS; Lissamine Rhodamine;Lissamine Rhodamine B; Calcein/Ethidium homodimer; LOLO-1; LO-PRO-1;Lucifer Yellow; Lyso Tracker Blue; Lyso Tracker Blue-White; Lyso TrackerGreen; Lyso Tracker Red; Lyso Tracker Yellow; LysoSensor Blue;LysoSensor Green; LysoSensor Yellow/Blue; Mag Green; Magdala Red(Phloxin B); Mag-Fura Red; Mag-Fura-2; Mag-Fura-5; Mag-Indo-1; MagnesiumGreen; Magnesium Orange; Malachite Green; Marina Blue; Maxilon BrilliantFlavin 10 GFF; Maxilon Brilliant Flavin 8 GFF; Merocyanin;Methoxycoumarin; Mitotracker Green FM; Mitotracker Orange; MitotrackerRed; Mitramycin; Monobromobimane; Monobromobimane (mBBr-GSH);Monochlorobimane; MPS (Methyl Green Pyronine Stilbene); NBD; NBD Amine;Nile Red; Nitrobenzoxadidole; Noradrenaline; Nuclear Fast Red; NuclearYellow; Nylosan Brilliant Iavin EBG; Oregon Green; Oregon Green 488-X;Oregon Green™; Oregon Green™ 488; Oregon Green™ 500; Oregon Green™ 514;Pacific Blue; Pararosaniline (Feulgen); PBFI; PE-Cy5; PE-Cy7; PerCP;PerCP-Cy5.5; PE-TexasRed [Red 613]; Phloxin B (Magdala Red); PhorwiteAR; Phorwite BKL; Phorwite Rev; Phorwite RPA; Phosphine 3R;Phycoerythrin B [PE]; Phycoerythrin R [PE]; PKH26 (Sigma); PKH67; PMIA;Pontochrome Blue Black; POPO-1; POPO-3; PO-PRO-1; PO-PRO-3; Primuline;Procion Yellow; Propidium Iodid (PI); PyMPO; Pyrene; Pyronine; PyronineB; Pyrozal Brilliant Flavin 7GF; QSY 7; Quinacrine Mustard; Red 613[PE-TexasRed]; Resorufin; RH 414; Rhod-2; Rhodamine; Rhodamine 110;Rhodamine 123; Rhodamine 5 GLD; Rhodamine 6G; Rhodamine B; Rhodamine B200; Rhodamine B extra; Rhodamine BB; Rhodamine BG; Rhodamine Green;Rhodamine Phallicidine; Rhodamine Phalloidine; Rhodamine Red; RhodamineWT; Rose Bengal; R-phycocyanine; R-phycoerythrin (PE); RsGFP; S65A;S65C; S65L; S65T; Sapphire GFP; SBFI; Serotonin; Sevron Brilliant Red2B; Sevron Brilliant Red 4G; Sevron Brilliant Red B; Sevron Orange;Sevron Yellow L; sgBFP™; sgBFP™ (super glow BFP); sgGFP™; sgGFP™ (superglow GFP); SITS; SITS (Primuline); SITS (Stilbene IsothiosulphonicAcid); SNAFL calcein; SNAFL-1; SNAFL-2; SNARF calcein; SNARF1; SodiumGreen; SpectrumAqua; SpectrumGreen; SpectrumOrange; Spectrum Red; SPQ(6-methoxy-N-(3-sulfopropyl)quinolinium); Stilbene; Sulphorhodamine Bcan C; Sulphorhodamine G Extra; SYTO 11; SYTO 12; SYTO 13; SYTO 14; SYTO15; SYTO 16; SYTO 17; SYTO 18; SYTO 20; SYTO 21; SYTO 22; SYTO 23; SYTO24; SYTO 25; SYTO 40; SYTO 41; SYTO 42; SYTO 43; SYTO 44; SYTO 45; SYTO59; SYTO 60; SYTO 61; SYTO 62; SYTO 63; SYTO 64; SYTO 80; SYTO 81; SYTO82; SYTO 83; SYTO 84; SYTO 85; SYTOX Blue; SYTOX Green; SYTOX Orange;Tetracycline; Tetramethylrhodamine (TRITC); Texas Red™; Texas Red-X™conjugate; Thiadicarbocyanine (DiSC3); Thiazine Red R; Thiazole Orange;Thioflavin 5; Thioflavin S; Thioflavin TCN; Thiolyte; Thiozole Orange;Tinopol CBS (Calcofluor White); TMR; TO-PRO-1; TO-PRO-3; TO-PRO-5;TOTO-1; TOTO-3; TriColor (PE-Cy5); TRITCTetramethylRodaminelsoThioCyanate; True Blue; TruRed; Ultralite; UranineB; Uvitex SFC; wt GFP; WW 781; X-Rhodamine; XRITC; Xylene Orange; Y66F;Y66H; Y66W; Yellow GFP; YFP; YO-PRO-1; YO-PRO-3; YOYO-1; and YOYO-3. Asused herein, a “fluorophore” may include a salt of the fluorophore.

Glucocerebrosidase Activity Modulation

The compounds disclosed herein preferably modulate activity ofglucocerebrosidase. Modulation may include inhibiting or decreasingglucocerebrosidase activity. Modulation also may include activating orincreasing glucocerebrosidase activity. Glucocerebrosidase activity maybe assessed utilizing methods known in the art and the methods disclosedherein, including the methods disclosed in the Examples provided herein.In some embodiments, the compounds decrease or increaseglucocerebrosidase activity relative to a control (e.g., by at leastabout 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%,400%, 500%, 600%, 700%, 800%, 900%, 1000% or more (or within a rangebounded by any of these values)). In other embodiments, the compoundsactivate glucocerebrosidase greater than about 2-fold, 3-fold, 4-fold,5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold,80-fold, 90-fold, or 100-fold, relative to a control. In otherembodiments, the compounds activate glucocerebrosidase with a maximumactivation (Eurax) greater than about 100%, 200%, 300%, 400%, 500%,600%, 700%, 800%, 900%, 1000%, 1100%, 1200%, 1300%, 1400%, or 1500% (orwithin a range bounded by any of these values). In other embodiments, anAC₅₀ value for the compound in regard to activation ofglucocerebrosidase may be determined and preferably the compound has anAC₅₀ value of less than about 10 μM, 5 μM, or 1 μM, 0.5 μM, 0.1 μM, 0.05μM, 0.01 μM, 0.005 μM, or 0.001 μM (or within a range bounded by any ofthese values).

Methods for measuring glucocerebrosidase activity are known in the art.(See, e.g., U.S. Publication No. 2017/0001976 and U.S. Publication No.2017/0002013 and Zheng et al. “Design and Synthesis of PotentQuinazolines as Selective β-Glucocerebrosidase Modulators, J. Med. Chem.2016 Sep 22; 59(18): 8508-8520; the contents of which are incorporatedherein by reference in their entireties).

Pharmaceutical Compositions and Methods of Administration

The compounds employed in the compositions and methods disclosed hereinmay be administered as pharmaceutical compositions and, therefore,pharmaceutical compositions incorporating the compounds are consideredto be embodiments of the compositions disclosed herein. Suchcompositions may take any physical form which is pharmaceuticallyacceptable; illustratively, they can be orally administeredpharmaceutical compositions. Such pharmaceutical compositions contain aneffective amount of a disclosed compound, which effective amount isrelated to the daily dose of the compound to be administered. Eachdosage unit may contain the daily dose of a given compound or eachdosage unit may contain a fraction of the daily dose, such as one-halfor one-third of the dose. The amount of each compound to be contained ineach dosage unit can depend, in part, on the identity of the particularcompound chosen for the therapy and other factors, such as theindication for which it is given. The pharmaceutical compositionsdisclosed herein may be formulated so as to provide quick, sustained, ordelayed release of the active ingredient after administration to thepatient by employing well known procedures.

The compounds for use according to the methods of disclosed herein maybe administered as a single compound or a combination of compounds. Forexample, a compound that modulates glucocerebrosidase activity may beadministered as a single compound or in combination with anothercompound that modulates glucocerebrosidase activity or that has adifferent pharmacological activity.

As indicated above, pharmaceutically acceptable salts of the compoundsare contemplated and also may be utilized in the disclosed methods. Theterm “pharmaceutically acceptable salt” as used herein, refers to saltsof the compounds which are substantially non-toxic to living organisms.Typical pharmaceutically acceptable salts include those salts preparedby reaction of the compounds as disclosed herein with a pharmaceuticallyacceptable mineral or organic acid or an organic or inorganic base. Suchsalts are known as acid addition and base addition salts. It will beappreciated by the skilled reader that most or all of the compounds asdisclosed herein are capable of forming salts and that the salt forms ofpharmaceuticals are commonly used, often because they are more readilycrystallized and purified than are the free acids or bases.

Acids commonly employed to form acid addition salts may includeinorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodicacid, sulfuric acid, phosphoric acid, and the like, and organic acidssuch as p-toluenesulfonic, methanesulfonic acid, oxalic acid,p-bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid,benzoic acid, acetic acid, and the like. Examples of suitablepharmaceutically acceptable salts may include the sulfate, pyrosulfate,bisulfate, sulfite, bisulfate, phosphate, monohydrogenphosphate,dihydrogenphosphate, metaphosphate, pyrophosphate, bromide, iodide,acetate, propionate, decanoate, caprylate, acrylate, formate,hydrochloride, dihydrochloride, isobutyrate, caproate, heptanoate,propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate,maleat-, butyne-1,4-dioate, hexyne-1,6-dioate, benzoate, chlorobenzoate,methylbenzoate, hydroxybenzoate, methoxybenzoate, phthalate,xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate,citrate, lactate, alpha-hydroxybutyrate, glycolate, tartrate,methanesulfonate, propanesulfonate, naphthalene-1-sulfonate,naphthalene-2-sulfonate, mandelate, and the like.

Base addition salts include those derived from inorganic bases, such asammonium or alkali or alkaline earth metal hydroxides, carbonates,bicarbonates, and the like. Bases useful in preparing such salts includesodium hydroxide, potassium hydroxide, ammonium hydroxide, potassiumcarbonate, sodium carbonate, sodium bicarbonate, potassium bicarbonate,calcium hydroxide, calcium carbonate, and the like.

The particular counter-ion forming a part of any salt of a compounddisclosed herein is may not be critical to the activity of the compound,so long as the salt as a whole is pharmacologically acceptable and aslong as the counterion does not contribute undesired qualities to thesalt as a whole. Undesired qualities may include undesirably solubilityor toxicity.

Pharmaceutically acceptable esters and amides of the compounds can alsobe employed in the compositions and methods disclosed herein. Examplesof suitable esters include alkyl, aryl, and aralkyl esters, such asmethyl esters, ethyl esters, propyl esters, dodecyl esters, benzylesters, and the like. Examples of suitable amides include unsubstitutedamides, monosubstituted amides, and disubstituted amides, such as methylamide, dimethyl amide, methyl ethyl amide, and the like.

In addition, the methods disclosed herein may be practiced using solvateforms of the compounds or salts, esters, and/or amides, thereof. Solvateforms may include ethanol solvates, hydrates, and the like.

The pharmaceutical compositions may be utilized in methods of treating adisease or disorder associated glucocerebrosidase activity. For example,the pharmaceutical compositions may be utilized to treat patients havingor at risk for acquiring Gaucher's disease and neurological diseases anddisorders such as genetic and sporadic synucleinopathies, includingParkinson's disease, dementia with Lewy bodies, and multiple systematrophy associated with aberrant glucocerebrosidase activity. Suitablepatients include, for example mammals, such as humans and non-humanprimates (e.g., chimps) or other mammals (e.g., dogs, cats, horses,rats, and mice). Suitable human patients may include, for example, thosewho have previously been determined to be at risk of having ordeveloping Gaucher's disease and neurological diseases and disorderssuch as genetic and sporadic synucleinopathies, including Parkinson'sdisease, dementia with Lewy bodies, and multiple system atrophyassociated with aberrant glucocerebrosidase activity.

As used herein, the terms “treating” or “to treat” each mean toalleviate symptoms, eliminate the causation of resultant symptoms eitheron a temporary or permanent basis, and/or to prevent or slow theappearance or to reverse the progression or severity of resultantsymptoms of the named disease or disorder. As such, the methodsdisclosed herein encompass both therapeutic and prophylacticadministration.

As used herein the term “effective amount” refers to the amount or doseof the compound, upon single or multiple dose administration to thesubject, which provides the desired effect in the subject underdiagnosis or treatment. The disclosed methods may include administeringan effective amount of the disclosed compounds (e.g., as present in apharmaceutical composition) for treating a disease or disorderassociated with glucocerebrosidase activity.

An effective amount can be readily determined by the attendingdiagnostician, as one skilled in the art, by the use of known techniquesand by observing results obtained under analogous circumstances. Indetermining the effective amount or dose of compound administered, anumber of factors can be considered by the attending diagnostician, suchas: the species of the subject; its size, age, and general health; thedegree of involvement or the severity of the disease or disorderinvolved; the response of the individual subject; the particularcompound administered; the mode of administration; the bioavailabilitycharacteristics of the preparation administered; the dose regimenselected; the use of concomitant medication; and other relevantcircumstances.

A typical daily dose may contain from about 0.01 mg/kg to about 100mg/kg (such as from about 0.05 mg/kg to about 50 mg/kg and/or from about0.1 mg/kg to about 25 mg/kg) of each compound used in the present methodof treatment.

Compositions can be formulated in a unit dosage form, each dosagecontaining from about 1 to about 500 mg of each compound individually orin a single unit dosage form, such as from about 5 to about 300 mg, fromabout 10 to about 100 mg, and/or about 25 mg. The term “unit dosageform” refers to a physically discrete unit suitable as unitary dosagesfor a patient, each unit containing a predetermined quantity of activematerial calculated to produce the desired therapeutic effect, inassociation with a suitable pharmaceutical carrier, diluent, orexcipient.

Oral administration is an illustrative route of administering thecompounds employed in the compositions and methods disclosed herein.Other illustrative routes of administration include transdermal,percutaneous, intravenous, intramuscular, intranasal, buccal,intrathecal, intracerebral, or intrarectal routes. The route ofadministration may be varied in any way, limited by the physicalproperties of the compounds being employed and the convenience of thesubject and the caregiver.

As one skilled in the art will appreciate, suitable formulations includethose that are suitable for more than one route of administration. Forexample, the formulation can be one that is suitable for bothintrathecal and intracerebral administration. Alternatively, suitableformulations include those that are suitable for only one route ofadministration as well as those that are suitable for one or more routesof administration, but not suitable for one or more other routes ofadministration. For example, the formulation can be one that is suitablefor oral, transdermal, percutaneous, intravenous, intramuscular,intranasal, buccal, and/or intrathecal administration but not suitablefor intracerebral administration.

The inert ingredients and manner of formulation of the pharmaceuticalcompositions are conventional. The usual methods of formulation used inpharmaceutical science may be used here. All of the usual types ofcompositions may be used, including tablets, chewable tablets, capsules,solutions, parenteral solutions, intranasal sprays or powders, troches,suppositories, transdermal patches, and suspensions. In general,compositions contain from about 0.5% to about 50% of the compound intotal, depending on the desired doses and the type of composition to beused. The amount of the compound, however, is best defined as the“effective amount”, that is, the amount of the compound which providesthe desired dose to the patient in need of such treatment. The activityof the compounds employed in the compositions and methods disclosedherein are not believed to depend greatly on the nature of thecomposition, and, therefore, the compositions can be chosen andformulated primarily or solely for convenience and economy.

Capsules are prepared by mixing the compound with a suitable diluent andfilling the proper amount of the mixture in capsules. The usual diluentsinclude inert powdered substances (such as starches), powdered cellulose(especially crystalline and microcrystalline cellulose), sugars (such asfructose, mannitol and sucrose), grain flours, and similar ediblepowders.

Tablets are prepared by direct compression, by wet granulation, or bydry granulation. Their formulations usually incorporate diluents,binders, lubricants, and disintegrators (in addition to the compounds).Typical diluents include, for example, various types of starch, lactose,mannitol, kaolin, calcium phosphate or sulfate, inorganic salts (such assodium chloride), and powdered sugar. Powdered cellulose derivatives canalso be used. Typical tablet binders include substances such as starch,gelatin, and sugars (e.g., lactose, fructose, glucose, and the like).Natural and synthetic gums can also be used, including acacia,alginates, methylcellulose, polyvinylpyrrolidine, and the like.Polyethylene glycol, ethylcellulose, and waxes can also serve asbinders.

Tablets can be coated with sugar, e.g., as a flavor enhancer andsealant. The compounds also may be formulated as chewable tablets, byusing large amounts of pleasant-tasting substances, such as mannitol, inthe formulation. Instantly dissolving tablet-like formulations can alsobe employed, for example, to assure that the patient consumes the dosageform and to avoid the difficulty that some patients experience inswallowing solid objects.

A lubricant can be used in the tablet formulation to prevent the tabletand punches from sticking in the die. The lubricant can be chosen fromsuch slippery solids as talc, magnesium and calcium stearate, stearicacid, and hydrogenated vegetable oils.

Tablets can also contain disintegrators. Disintegrators are substancesthat swell when wetted to break up the tablet and release the compound.They include starches, clays, celluloses, algins, and gums. As furtherillustration, corn and potato starches, methylcellulose, agar,bentonite, wood cellulose, powdered natural sponge, cation-exchangeresins, alginic acid, guar gum, citrus pulp, sodium lauryl sulfate, andcarboxymethylcellulose can be used.

Compositions can be formulated as enteric formulations, for example, toprotect the active ingredient from the strongly acid contents of thestomach. Such formulations can be created by coating a solid dosage formwith a film of a polymer which is insoluble in acid environments andsoluble in basic environments. Illustrative films include celluloseacetate phthalate, polyvinyl acetate phthalate, hydroxypropylmethylcellulose phthalate, and hydroxypropyl methylcellulose acetatesuccinate.

When it is desired to administer the compound as a suppository,conventional bases can be used. Illustratively, cocoa butter is atraditional suppository base. The cocoa butter can be modified byaddition of waxes to raise its melting point slightly. Water-misciblesuppository bases, such as polyethylene glycols of various molecularweights, can also be used in suppository formulations.

Transdermal patches can also be used to deliver the compounds.Transdermal patches can include a resinous composition in which thecompound will dissolve or partially dissolve; and a film which protectsthe composition and which holds the resinous composition in contact withthe skin. Other, more complicated patch compositions can also be used,such as those having a membrane pierced with a plurality of poresthrough which the drugs are pumped by osmotic action.

As one skilled in the art will also appreciate, the formulation can beprepared with materials (e.g., actives excipients, carriers (such ascyclodextrins), diluents, etc.) having properties (e.g., purity) thatrender the formulation suitable for administration to humans.Alternatively, the formulation can be prepared with materials havingpurity and/or other properties that render the formulation suitable foradministration to non-human subjects, but not suitable foradministration to humans.

The compounds disclosed in the present application may function asactivators of glucocerebrosidase. For example, a compound disclosedherein may be reacted with glucocerebrosidase to prepare an activatedglucocerebrosidase that is covalent attached to the compound. Theactivated glucocerebrosidase thusly formed may be prepared as apharmaceutical composition to treat and/or prevent a disease or disorderthat is associated with glucocerebrosidase activity as in enzymereplacement therapy, which is known in the art.

The following list of formulations is illustrative. These illustrativeformulations may be suitable for preparing pharmaceutical compositionsthat include the disclosed compounds as “active ingredients.” Thefollowing list of formulations is illustrative and should not beinterpreted as limiting the present disclosure or claims in any way:

Formulation 1

Hard gelatin capsules are prepared using the following ingredients:

Quantity (mg/capsule) Active Ingredient 250 Starch, dried 200 Magnesiumstearate  10 Total 460 mg

The above ingredients are mixed and filled into hard gelatin capsules in460 mg quantities.

Formulation 2

Quantity (mg/tablet) Active Ingredient 250 Cellulose, microcrystalline400 Silicon dioxide, fumed  10 Stearic acid  5 Total 665 mg

The components are blended and compressed to form tablets each weighing665 mg.

Formulation 3

An aerosol solution is prepared containing the following components:

Weight % Active Ingredient  0.25 Ethanol  29.75 Propellant 22(chlorodifluoromethane)  70.00 Total 100.00

The active compound is mixed with ethanol and the mixture added to aportion of the Propellant 22, cooled to −30° C. and transferred to afilling device. The required amount is then fed to a stainless steelcontainer and diluted with the remainder of the propellant. The valveunits are then fitted to the container.

Formulation 4

Tablets each containing 60 mg of active ingredient are made as follows:

Active Ingredient  60 mg Starch  45 mg Microcrystalline cellulose  35 mgPolyvinylpyrrolidone   4 mg Sodium carboxymethyl starch 4.5 mg Magnesiumstearate 0.5 mg Talc   1 mg Total 150 mg

The active ingredient, starch, and cellulose are passed through a No. 45mesh U.S. sieve and mixed thoroughly. The solution ofpolyvinylpyrrolidone is mixed with the resultant powders which are thenpassed through a No. 14 mesh U.S. sieve. The granules so produced aredried at 50° C. and passed through a No. 18 mesh U.S. sieve. The sodiumcarboxymethyl starch, magnesium stearate, and talc, previously passedthrough a No. 60 mesh U.S. sieve, are then added to the granules which,after mixing, are compressed on a tablet machine to yield tablets eachweighing 150 mg.

Formulation 5

Capsules, each containing 80 mg medicament, are made as follows:

Active Ingredient  80 mg Starch  59 mg Microcrystalline cellulose  59 mgMagnesium stearate  2 mg Total 200 mg

The active ingredient, cellulose, starch, and magnesium stearate areblended, passed through a No. 45 sieve, and filled into hard gelatincapsules in 200 mg quantities.

Formulation 6

Suppositories each containing 225 mg of active ingredient may be made asfollows:

Active Ingredient   225 mg Saturated fatty acid glycerides 2,000 mgTotal 2,225 mg

The active ingredient is passed through a No. 60 mesh U.S. sieve andsuspended in the saturated fatty acid glycerides previously melted usingthe minimum heat necessary. The mixture is then poured into asuppository mold of nominal 2 g capacity and allowed to cool.

Formulation 7

Suspensions each containing 50 mg of medicament per 5 ml dose are madeas follows:

Active Ingredient   50 mg Sodium carboxymethyl cellulose   50 mg Syrup1.25 ml Benzoic acid solution 0.10 ml Flavor q.v. Color q.v. Purifiedwater to total   5 ml

The medicament is passed through a No. 45 mesh U.S. sieve and mixed withthe sodium carboxymethyl, cellulose and syrup to form a smooth paste.The benzoic acid solution, flavor, and color are diluted with some ofthe water and added with stirring. Sufficient water is then added toproduce the required volume.

Formulation 8

An intravenous formulation containing 100 mg of medicament per 5 ml dosecan be prepared as follows:

Active Ingredient 100 mg Mannitol 100 mg 5 N Sodium hydroxide 200 mlPurified water to total  5 ml

EXAMPLES

The followings Examples are illustrative only and are not intended tolimit the scope of the claimed subject matter.

Pyrrolopyrimidine Compounds as Glucocerebrosidase Modulators and TheirApplications

Technical Field

The technical field relates to the development of a new series ofpyrrolopyrimidine glucocerebrosidase (GCase) activators and acompound-activated form of GCase implicated in Gaucher's disease andParkinson's disease, and the activated GCase for high throughputscreening of compounds that modulate the activity of GCase.

Abstract

Gaucher's disease is a rare genetic disease caused by GBA1(glucocerebrosidase) gene mutations. Currently, the treatment for Type 1Gaucher's disease is enzyme replacement therapy (ERT) administered everytwo weeks. ERT is very expensive and not effective for neuropathic formsof Gaucher's disease. Mutations in GBA1 are also linked to Parkinson'sdisease (PD), increasing the risk of PD. The pharmacological chaperonestrategy to activate GCase has been previously attempted, but, none ofthe compounds achieved success in clinical trials because they targetedthe active site of GCase. In contrast, our novel pyrrolopyrimidinecompounds produce high activity of GCase. In addition, covalent bindingpyrrolopyrimidine compounds activated wild-type GCase up to 70-100 foldin efficiency. We also found that the active enzyme is more stable inacidic environment. Moreover, we found that the activated enzyme couldbe inhibited with the same site binding modulators, suggesting itsutility in high throughput screening of novel GCase modulators.

Applications

Applications of the disclosed technology include, but are not limitedto: (i) prevention or treatment of Gaucher's disease andneurodegenerative diseases characterized by neuronal degradation ordamage, including but not limited to Parkinson's disease via activationof glucocerebrosidase (GCase); (ii) preparation of an activated GCasethat can be used as a new form of enzyme replacement therapy, includingbut not limited to Gaucher's disease; (iii) new compounds forglucocerebrosidase activation in synucleinopathies; and (iv) preparationof an activated enzyme that can be used for applications including butnot limited to high throughput screening, testing of the bindingaffinity of compounds for activated GCase.

Advantages

Advantages of the disclosed technology include, but are not limited to:(i) the disclosed pyrrolopyrimidine-based compounds have improved enzymeactivity and binding affinity with GCase and have better chemical andphysical properties (such as polar surface area, molecular weight,solubility, CLogP, number of rotatable bonds, and number of hydrogenbonds) over previous GCase activators, and these properties are closerto those required for oral bioavailability and blood-brain barrierpenetration; (ii) the disclosed pyrrolopyrimidine compound-activatedGCase is more active in vitro and stable in acidic buffer than wild typeGCase, and therefore, when used for enzyme replacement therapy, theenzyme amount and frequency could be reduced; and (iii) the disclosedactivated enzyme can be used in enzyme activity assay, which is a veryeasy and quick method to test the binding affinity of the compounds withGCase; and (iv) the disclosed enzyme activity assay is very useful tofind out new compounds binding with GCase, where no high throughputscreening method based on a binding affinity assay had previously beendeveloped and disclosed in the art.

Brief Summary of the Technology

Here, we disclose a new class of chemical compounds (substitutedpyrrolopyrimidines), which are activators of glucocerebrosidase (GCase).Such compounds could be used for both the study of this enzyme andpotentially for development into drug candidates for treatment orprevention of Gaucher's disease and neurodegenerative disorders. Acompound activated enzyme showed much more activity and stability andcan be used for Gaucher's disease and neurodegenerative disorders. Theactivated enzyme can be used in an enzyme activity assay, which is avery easy and quick method to test the binding affinity of the compoundswith GCase. Further details are provided in the attached report.

Related Publications

Ehud Goldin, Juan Jose Marugan, Omid Motabar, Samarjit Patnaik, EllenSidransky, Noel Southall, Wendy Westbrook. Substitutedpyrazolopyrimidines as glucocerebrosidase activators. December 2010,WO2012078855 A1.

Patnaik S1, Zheng W, Choi J H, Motabar O, Southall N, Westbroek W, Lea WA, Velayati A, Goldin E, Sidransky E, Leister W, Marugan J J. Discovery,structure-activity relationship, and biological evaluation ofnoninhibitory small molecule chaperones of glucocerebrosidase. J MedChem. 2012; 55(12): 5734-48.

Krainc D., Silverman R B., and Zheng, J. Substituted4-Methyl-Pyrrolo[1,2-a]Pyrimidine-8-carboxamide compounds and usesthereof for modulating glucocerebrosidase activity, Oct., 31, 2017. U.S.Pat. No. 9,802,942.

Summary

Gaucher's disease is a rare genetic disease caused by GBA1(glucocerebrosidase) gene mutations. Currently, the treatment for Type 1Gaucher's disease is enzyme replacement therapy (ERT), administeredevery two weeks. ERT is very expensive and not effective for neuropathicforms of Gaucher's disease. Mutations in GBA1 are also linked toParkinson's disease (PD), increasing the risk of PD. The pharmacologicalchaperone strategy to activate GCase has been previously attempted, but,none of the compounds achieved success in clinical trials because theytargeted the active site of GCase. In contrast, our novelpyrrolopyrimidine compounds produced high activity of GCase. Inaddition, covalent binding pyrrolopyrimidine compounds activatedwild-type GCase up to 70-100 fold in efficiency. We also found that theactive enzyme is more stable in an acidic environment, like thelysosome. Moreover, we found that the activated enzyme activity could beinhibited by competitive modulators in an enzyme activity assay,suggesting their utility in high throughput screening of novel GCasemodulators.

Experimental

The disclosed pyrrolopyrimidine compounds were synthesized and testedusing the methods described below. Additional methods for synthesizingand testing pyrrolopyrimidine compounds are described in the art. (See,e.g., Zheng et al., β-Glucocerebrosidase Modulators Promote Dimerizationof β-Glucocerebrosidase and Reveal an Allosteric Binding Site, J. Am.Chem. Soc., 2018, 140 (18), pp 5914-5924; and U.S. Pat. No. 9,802,942;the contents of which are incorporated herein by reference in theirentireties).

Preparation of 2,5-dioxopyrrolidin-1-yl2-(3-(2,4-dimethylpyrrolo[1,2-a]pyrimidine-8-carboxamido)phenyl)acetate(3)

Preparation of methyl 2-(3-(2,4-dimethylpyrrolo[1,2-a]pyrimidine-8-carboxamido)phenyl)acetate (1).2,4-Dimethylpyrrolo[1,2-a]pyrimidine-8-carboxylic acid (95 mg, 0.5mmol), HATU (190 mg, 0.5 mmol), and diisopropylethylamine (350 μL) weredissolved in DMF (3 mL), and stirred for 30 min at room temperature.Methyl 2-(3-aminophenyl)acetate (83 mg, 0.5 mmol) was added into thereaction mixture. The mixture was allowed to stirred at 50° C. for 5h.The mixture was cooled to room temperature, and diluted with water andfiltered. The residue was washed with water (×2) and dried under vacuumto obtain a yellow solid (147 mg, 70%).

Preparation of 2-(3-(2,4-dimethylpyrrolo[1,2-a]pyrimidine-8-carboxamido)phenyl)acetic acid(2). To a solution of 4N NaOH (4 mL) and MeOH (4 mL) was added methyl2-(3-(2,4-dimethylpyrrolo[1,2-a]pyrimidine-8-carboxamido)phenyl)acetate(1) (100 mg, 0.3 mmol), and the mixture was stirred at 50° C. overnight.The mixture was cooled to room temperature, and adjusted with 1N HCl topH 1. The formed solid was filtered, washed with water (×2), and driedunder vacuum to obtain a yellow solid (79 mg, 82%).

Preparation of 2,5-dioxopyrrolidin-1-yl 2-(3-(2,4-dimethylpyrrolo[1,2-a]pyrimidine-8-carboxamido)phenyl)acetate (3).A mixture of2-(3-(2,4-dimethylpyrrolo[1,2-a]pyrimidine-8-carboxamido)phenyl)aceticacid (2) (31 mg, 0.1 mmol), N-Hydroxysuccinimide (11.5 mg, 0.1 mmol),EDCI (19 mg, 0.1 mmol), and triethylamine (15 μL) in DCM (2 mL) wasstirred at 50° C. for 4h. The mixture was diluted with DCM (20 mL), andwashed with water (5 mL). The organic layer was washed with brine, dried(Na₂SO₄), concentrated. The residue was purified with flashchromatography to give yellow solid (25 mg, 60%).

¹H NMR (500 MHz, CDCl₃) δ 10.74 (s, 1H), 7.73 (d, J=9.0 Hz, 2H), 7.55(d, J=3.2 Hz, 1H), 7.33 (t, J=7.8 Hz, 1H), 7.05 (d, J=3.5 Hz, 2H), 6.50(s, 1H), 3.95 (s, 2H), 2.81 (s, 4H), 2.62 (s, 3H), 2.56 (s, 3H). ¹³C NMR(125 MHz, CDCl₃) δ 168.9, 166.7, 162.4, 155.5, 142.5, 140.0, 139.4,131.9, 129.3, 123.6, 120.3, 118.9, 117.2, 108.3, 106.9, 105.7, 37.7,25.5, 24.7, 18.1. HRMS (ESI): calcd for C₂₂H₂₁N₄O₅ [M+H]⁺, 421.1506;found, 421.1506.

GCase Covalent Activation Assay with Compound 3. To wild-type GCase (22μM, 1 mL, 1 equiv) in PBS (pH 7.4) was added covalent compound 3 in DMSO(0.89 mM, 50 μL, 2 equiv) in one portion and immediately vortexed for 5sec. The reaction mixture was shaken at room temperature for 2 h, andanother portion (25 μL, 1 equiv) of compound 3 was added, and reactedfor 2 h. The reaction process was monitored by high resolution LC-MSSpectra. Then, the mixture was dialyzed by repeated buffer exchangeswith 0.1 M acetic acid/sodium acetate buffer (pH 5.0) using Amicon Ultra0.5 ml 10K centrifugal filters (Millipore). The concentrated enzyme waspurified by size exclusion chromatography (Superdex 200 column, GEHealthcare Life Sciences), using 0.1 M acetic acid/sodium acetate buffer(pH 5.0) as the elution solvent. The purified enzyme was assayed withRes-β-Glc and 4MU-β-Glc substrates.

High Throughput Screen with Compound 6 Covalently Activated GCase. Thecompounds stock (10 mM) in DMSO solution (50 nL/well) were transferredto a black 384-well plate (25 μM). The compound 3 activated enzymesolution (10 μL, 2 nM final concentration) was transferred to the wells.After 5 min of incubation at room temperature, the enzyme reaction wasinitiated by the addition of blue substrate (4MU-β-Glc) (10 μL/well).The final concentration of the blue substrate was 1.5 mM. The bluesubstrate reaction was terminated by the addition of 10 μL/well stopsolution (1 M NaOH and 1 M glycine mixture, pH 10) after 30 min ofincubation at room temperature. The fluorescence was then measured in aBiotek Synergy H1 multi-mode plate reader with λ_(ex)=365 nm andλ_(cm)=440 nm.

TABLE 1 Exemplary Compounds and Activities Maximum AC₅₀ Activation No.Structures (μM) (Emax) (%)  1

6.59  450  2

30  550  3

9.09  500  4

1.18  400  5

2.87  175  6

4.59  460  7

3.39  210  8

3.43  480  9

2.23  280 10

0.77  202 11

1.41  400 12

3.44  300 13

0.63  180 14

6.46 1100 15

36.58  700 16

1.49  225 17

3.88  750 18

NA 19

0.71  250 20

6.10  550 21

35.48  622 22

35.48  429 23

28.2  644 24

3.55  527 25

7.94  456 26

1.12  290 27

2.82  620 28

50  115 29

4.47  374 30

1.12  198 31

1.00  355 32

NA 33

39.8  914 34

3.55  578 35

2.24  298 36

1.42  280 37

8.91  427 38

4.47  810 39

10 1446 40

4.47 1366 41

28.18 1187 42

5.0  715 43

1.41  296 44

1.58  342 45

1.78  325 46

1.12  230 47

NA 48

3.16  796 49

3.55 1150 50

4.47  230 51

10  172

It will be readily apparent to one skilled in the art that varyingsubstitutions and modifications may be made to the invention disclosedherein without departing from the scope and spirit of the invention. Theinvention illustratively described herein suitably may be practiced inthe absence of any element or elements, limitation or limitations whichis not specifically disclosed herein. The terms and expressions whichhave been employed are used as terms of description and not oflimitation, and there is no intention in the use of such terms andexpressions of excluding any equivalents of the features shown anddescribed or portions thereof, but it is recognized that variousmodifications are possible within the scope of the invention. Thus, itshould be understood that although the present invention has beenillustrated by specific embodiments and optional features, modificationand/or variation of the concepts herein disclosed may be resorted to bythose skilled in the art, and that such modifications and variations areconsidered to be within the scope of this invention.

Citations to a number of patent and non-patent references are madeherein. The cited references are incorporated by reference herein intheir entireties. In the event that there is an inconsistency between adefinition of a term in the specification as compared to a definition ofthe term in a cited reference, the term should be interpreted based onthe definition in the specification.

We claim:
 1. A compound or a salt or solvate thereof having a Formula I:

wherein: m is 0-6; R¹ is selected from

n is 0-6; p is 0-6; R² is C1-C6 alkyl or pyridinyl; R³ is selected fromhydrogen, C1-C6 alkyl, C1-C6 alkoxy, phenyl, nitrophenyl, phenoxy,amino, —C(O)—O—R¹², —N—C(O)—R¹³, and

R⁴ is —(OCH₂CH₂)_(q)— where q is 0-6; R⁵ is hydrogen, C1-C6 alkyl, orC3-C6 cycloalkyl; R⁶ and R⁷ are independent selected from hydrogen andC1-C6 alkyl; R⁸ is hydrogen, C1-C6 alkyl, phenyl, or benzyl; R⁹ ishydrogen, C1-C6 alkyl, or C1-C6 dialkyl; R¹⁰ and R¹¹ are independentlyselected from hydrogen, C1-C6 alkyl, phenyl, and benzyl; R¹² is selectedfrom hydrogen, C1-C6 alkyl, phenyl, and N-succinimidyl; and R¹³ isselected from hydrogen, C1-C6 alkyl, C1-C6 (halo)alkyl, and C2-C6alkenyl.
 2. The compound of claim 1 having Formula Ia:


3. The compound of claim 1 having a formula:


4. The compound of claim 1 having Formula Ib:


5. The compound of claim 1 having a formula selected from:


6. The compound of claim 1, wherein R³ is selected from —C(O)—O—R¹² and—N—C(O)—R¹³.
 7. The compound of claim 1 having formula:


8. The compound of claim 1 having formula:


9. A compound having a formula of any compound of Table
 1. 10. Apharmaceutical composition comprising the compound of claim 1 and apharmaceutical carrier.
 11. A method for treating a disease or disorderthat is associated with glucocerebrosidase activity in a subject in needthereof, the method comprising administering to the subject thecomposition of claim
 10. 12. The method of claim 11, wherein the diseaseor disorder is a degenerative neurological disease or disorder.
 13. Themethod of claim 12, wherein the degenerative neurological disease ordisorder is Gaucher's disease or Parkinson's disease.
 14. The compoundof claim 1 covalently attached to glucocerebrosidase.
 15. Apharmaceutical composition comprising: (i) the compound of claim 1covalently attached to glucocerebrosidase; and (ii) a pharmaceuticalcarrier.
 16. A method for treating a disease or disorder that isassociated with glucocerebrosidase activity in a subject in needthereof, the method comprising administering to the subject thecomposition of claim
 15. 17. The method of claim 16, wherein the diseaseor disorder is a degenerative neurological disease or disorder.
 18. Themethod of claim 17, wherein the degenerative neurological disease ordisorder is Gaucher's disease or Parkinson's disease.
 19. A method ofscreening for a test compound having binding activity or modulatoryactivity for glucocerebrosidase, the method comprising: (a) contactingthe test compound with an activated glucocerebrosidase that has beenactivated by covalently attaching to the glucocerebrosidase a compoundaccording to claim 1 and (b) detecting enzyme activity of the activatedglucocerebrosidase in the presence of the test compound.
 20. The methodof claim 19, wherein detecting enzyme activity comprises (i) contactingthe activated glucocerebrosidase and the test compound with a substratethat is metabolized by the activated glucocerebrosidase to generate afluorescent metabolite; and (ii) detecting fluorescence of thefluorescent metabolite.